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Ann Thorac Surg 2006;82:996-1002
© 2006 The Society of Thoracic Surgeons
a Department of Surgery, University of Minnesota Medical School, Minneapolis, Minnesota
b Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota
Accepted for publication April 3, 2006.
* Address correspondence to Dr Kratzke, Division of Hematology, Oncology, and Transplant, Department of Medicine, MMC 420, 480 Delaware St. SE, Minneapolis, MN 55455 (Email: kratz003{at}umn.edu).
Presented at the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
| The Thoracic Surgery Directors Association (TSDA) Resident Research Award was established in 1990 to encourage resident research in cardiothoracic surgery. Abstracts submitted to The Society of Thoracic Surgeons (STS) Program Committee representing research performed by residents were forwarded to the TSDA to be considered for this award. The abstracts were selected by the TSDA Executive Committee consisting of Jeffrey Gold, MD, President, John Brown, MD, President-Elect, John Calhoon, MD, Secretary/Treasurer, Douglas Mathisen, MD, Immediate Past President, George Hicks, MD, Councillor-at-Large, Bartley Griffith, MD, Councillor-at-Large, and Leslie Kohman, MD, Councillor-at-Large. The TSDA Resident Research Award was given to Bryan A. Whitson, MD, a resident at the University of Minnesota Department of Surgery Section of Thoracic Surgery and Ryan R. Davies, MD, a resident of New York-Presbyterian Hospital (Columbia University Medical Center). They each received a monetary award of $1,000.00 and an engraved desktop award. The TSDA makes this award annually. The resident author of the selected study is recognized at the STS meeting.
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Abstract |
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METHODS: Malignant mesothelioma cell lines, H2373 and H2461, previously shown to activate the IGF pathway were grown in culture. The adherent cells, initially plated at 5 x 105 cells/plate, were treated for 72 hours, in triplicate, with varying concentration of NVP-AEW541 (0, 1, 5, 10, 20, and 50 µM). Viable cells were counted every 24 hours. Additionally, separate cultures in serum-free medium were treated with NVP-AEW541, then stimulated with IGF and lysates collected for immunoblot analysis.
RESULTS: In both cell lines, 0, 1, 5, and 10 µM showed an inhibitory or static effect, while 20 and 50 µM were cidal. Immunoblot analysis demonstrated that phosphorylation of IGF-1R was inhibited by NVP-AEW541 at higher concentration. Phosphorylation of mitogen-activated protein kinase and Akt, downstream IGF pathway mediators, were also shown to be repressed by drug treatment.
CONCLUSIONS: NVP-AEW541 has a concentration-dependent inhibitory effect on mesothelioma cells in culture. NVP-AEW541 acts by inhibition of IGF-1R phosphorylation. Inhibition effects phosphorylation of downstream mediators in a dose-dependent fashion. Inhibition of the IGF pathway decreases viability of mesothelioma cell culture. Further evaluation of NVP-AEW541, and other selective IGF-1R inhibitors, may play an important role in multimodal treatment of malignant mesothelioma.
Malignant pleural mesothelioma (MPM) is diagnosed in approximately 3,000 new patients annually. The survival for these patients with currently available therapy is poor. Thus, newer targeted chemotherapeutic agents are sought for the treatment of this disease. One such potential agent is NVP-AEW541, a small molecule receptor tyrosine kinase inhibitor. This drug is targeted at the insulin-like growth factor type 1 receptor (IGF-1R). The IGF-1R pathway has been implicated in the tumorigenesis of breast, colon, and prostate cancer [1]. The IGF-1R activation is also implicated in MPM as it is quite uniformly found to be overexpressed in this disease [2, 3]. Work from this laboratory has previously shown that IGF-1R is overexpressed in 10 cell lines and 4 tumor samples [[3]. Other investigators have demonstrated IGF-1R overexpression in 6 other cell lines [2].
Previous work in MPM through the use of cDNA microarray has led to the identification of genes which are differentially expressed in MPM cell lines. For example, two such genes are the adaptor proteins and insulin receptor substrate (IRS) -1 and -2 [4]. Overexpression of these proteins is associated with increased proliferation and increased motility [4]. Two MPM cell lines previously demonstrated to express these proteins are NCI-H2373 (IRS-1) and NCI-H2461 (IRS-2).
Our primary aim was to evaluate the effect of treatment of the MPM cell lines NCI-H2373 and NCI-H2461 with the IGF-1R inhibitor NVP-AEW541 to demonstrate any growth inhibitory effects. Furthermore, we evaluated the effect of the NVP-AEW541 treatment on the activation of the IGF-1 receptor and its downstream mediators, mitogen-activated protein kinase (MAPK) and Akt.
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Material and Methods |
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Cell Inhibition
The NVP-AEW541 was a kind gift from Novartis (Novartis Pharma, Inc, Basel, Switzerland). Both cell lines were subjected to seven different treatment groups. There were six increasing concentrations of NVP-AEW541 (0, 1, 5, 10, 20, and 50 µM) diluted in culture media. There was one group of 0.1% dimethyl sulfoxide (DMSO) in media to correlate with the highest concentration of carrier in the NVP-AEW541 group (50 µM). Each cell line was plated on a 6-well plate (Becton Dickinson, Bedford, MA) at a concentration of 500,000 cells per well in 2.5 cm3 of appropriate group media. Cell culture was continued for 72 hours and every 24 hours the media was exchanged with new media and fresh drug at the appropriate treatment group concentration. Every 24 hours, 3 samples from each group (9 samples per group total) were washed twice with warm phosphate buffered saline (PBS) and then trypsinized (0.5 cm3). Cell count was then performed by 0.4% trypan blue exclusion on a hemacytometer. Statistical analysis was performed using SPSS for Windows, version 11 (SPSS, Inc, Chicago, IL). Groupings were compared by the pairwise Student t test with the 0 µM group being the reference for each cell line. A p value less than 0.05 was considered statistically significant.
Cell Stimulation and Lysate Preparation
Cells were grown in 10 cm dishes in regular growth media. Confluent cells (70%) were washed twice with warm PBS and then incubated in serum free medium (SFM) for 24 hours. The SFM consisted of improved MEM with zinc (Richter's modification) plus supplements (20 mM N-[2-hydroxyethyl]piperazine-N'=[4-butanesulfonic acid], 10 µL/mL trace elements, 2 µg/mL 5% transferrin, and 2 mg/mL fibronectin) [4]. After 24 hours, the media was removed and replaced with fresh SFM with the varying level of drug concentration. After 20 minutes, 5 nM IGF was added to the appropriate plates. At the completion of 30 minutes total incubation, the cells were washed twice with ice cold PBS and lysed with 500 µL/plate TNESV (50 mM Tris-Cl (pH 7.4), 1% NP40, 2 mM ethylenediaminetetraacetic acid (pH 8.0), 100 mM NaCl, 10 mM Na-orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/mL leupeptin, and 20 µg/mL aprotinin) [4]. Lysates were centrifuged at 4°C, supernatant collected, and placed in –80°C freezer. Protein concentrations were determined using Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc, Hercules, CA).
Immunoblotting
For immunoblotting, 50 µg of protein were resolved on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and proteins were transferred to polyvinylidene difluoride membrane. Membranes were blocked with 2% w/v enhanced chemiluminescence Advance blocking agent (Amersham Pharmacia Biotech, Piscataway, NJ) in tris-buffered saline (TBS) with 1% Tween 20 (TBS-T) overnight at 4°C, washed in TBS-T 5x 5 minutes, incubated with appropriate primary antibody for 1 hour at room temperature, and then washed 2x15 minutes and 3x 5 minutes with TBS-T. Subsequently, the membranes were incubated with secondary antibody for 1 hour at room temperature and then washed 2x 15 minutes and 3x 5 minutes with TBS-T. Chemiluminescence was performed using ECL Advance (Amersham Bioscience). For each phosphorylated protein, the unphosphorylated protein served as an internal loading control. For the IGF1Rß (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), p-IGF1R (Biosource International, Camarillo, CA), MAPK (Cell Signaling Technology, Danvers, MA), p-MAPK (Cell Signaling Technology), Akt (Cell Signaling Technology), and p-Akt (Cell Signaling Technology) immunoblots, the primary antibody was a 1:10,000 dilution and the secondary antibody was a 1:50,000 dilution of antirabbit conjugated horseradish peroxidase. For the p-epidermal growth factor receptor (p-EGFR) (Cell Signaling Technology) and EGFR (Cell Signaling Technology) immunoblots, ECL Plus (Amersham Bioscience) utilizing a primary antibody dilution of 1:500 and 1:1000, respectively, and secondary antibody at 1:5000 dilution of antirabbit conjugated horseradish peroxidase, were used.
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In those tumors where there is over expression of IGF-1R and its stimulation by IGF leads to activation of antiapoptotic pathways, such as MAPK and Akt. This pathway begins with IGF-1R phosphorylation on the cell membrane and continues by IRS-1 and IRS-2 activation intracellularly. Subsequently the pathway is mediated by the PI3K/Akt and MAPK subpathways. Akt prevents apoptosis by increased translation and an inhibition of cell cycle arrest [9, 10]. Similarly, the MAPK pathway (including the components MAPKK, MEK, MEKK, and MKK) increases gene expression and proliferation [10, 11]. The net sum enhancement of the malignant transformation is highlighted by inhibited apoptosis and increased proliferation. The interaction of the pathway components with the IGF-1 receptor is shown graphically in Figure 8. The IRS-2 activation from IGF stimulation has been associated with an increase in cellular motility [6].
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The NVP-AEW541 treatment of in vitro murine transformed fibroblast culture resulted in a dose-dependent IGF-1R phosphorylation inhibition [12]. The same researchers, in an in vivo murine fibrosarcoma model, were able to demonstrate a similar IGF-1R phosphorylation effect as well as tumor growth inhibition [12]. A similar effect has been demonstrated in solid malignancies as well. Upon treatment with NVP-AEW541, cell culture of Ewing's sarcoma, osteosarcoma, and rhabdosarcoma have growth inhibition and an increase in apoptosis [13]. The drug treatment was shown to have an effect by inhibiting phosphorylation of the IGF-1 receptor during IGF stimulation. The presumption is that downstream mediators are not being stimulated by the phosphorylation inhibition of the drug.
In MPM, the IGF-1R receptor level has been shown to be elevated in multiple cell lines, compared with normal tissue [2–4]. The potential application of a novel small molecule inhibitor in the multimodal treatment of MPM is of great interest. Our aim was then to evaluate if there was a growth inhibitory effect of NVP-AEW541 on MPM. Additionally, we aimed to elucidate if any inhibitory changes seen were due to effects on the downstream pathway mediators of the IGF pathway, namely Akt and MAPK.
Previous work from this laboratory utilizing cDNA microarray, immunoblotting, and immunoprecipitation has demonstrated that the IRS-1 and IRS-2 proteins are differentially activated in mesothelioma cell lines after IGF stimulation [4]. From this work, we chose one cell line that predominantly activates the IRS-1 subtype (associated with increased proliferation), NCI-H2373, and one cell line that predominantly activates the IRS-2 subtype (associated with increased migration).
Cell culture treatment with NVP-AEW541 convincingly demonstrates a dose-dependent inhibition effect (Figs 1; 2) in cell growth or cell proliferation. This effect was similar in both cell lines and at lower drug levels (ie, 1 to 10 µM) there was a static inhibitory effect while at higher drug levels (ie, 20 to 50 µM) there was a cidal inhibitory effect. These results mirror results found in other malignancies [12, 13].
Immunoblot analysis of IGF-1R in both NCI-H2373 and NCI-H2461 (Fig 3) demonstrate that with NVP-AEW541 treatment there is no decrease in the expression of the IGF-1R protein. In both cell lines, treatment with the drug repressed phosphorylation of IGF-1R in a dose-dependent manner. These effects on the P-IGF-1R and IGR-1R are similar to those obtained previously in solid malignancies [12, 13]. A potential concern with the compound is that there would be global nonspecific inhibition of other growth factor receptors. The immunoblots of EGFR and the P-EGFR seen in Figure 4 demonstrate that there is no inhibition of the EGFR and no reduction in the P-EGFR expression with increasing drug concentrations in the 2373 cell line and slight reduction in the 2461 cell line. This illustrates minimal nonspecific binding of the compound.
In the evaluation of the MAPK pathway, we were able to demonstrate that there was no expression change in MAP kinase during drug treatment. The P-MAPK immunoblots (Fig 5) reveal that phosphorylation of MAPK decreases increasing drug treatment concentrations. This portion of the pathway is associated with the increasing in translation and expression. Its inhibition likely results in decrease of translation.
When we directed our attention to the Akt pathway, similar results were obtained. Again, the Akt pathway activation is associated with an inhibition of apoptosis [9, 10]. The immunoblots for Akt in Figure 6 demonstrate that NVP-AEW541 treatment does not effect Akt protein level. The increasing drug treatment does inhibit Akt phosphorylation with even small drug concentrations.
The NVP-AEW541 treatment exhibits a dose-dependent inhibition of IGF-1 receptor activation through phosphorylation, which results in an inhibitory effect on the downstream mediators of the pathway, MAPK and Akt. The effects of increasing drug concentration in cell culture quite strikingly demonstrate a dose-dependent killing of two different cell lines, NCI-H2373 and NCH-H2461. These cell lines preferentially activate the IRS-1 and IRS-2, respectively. The same effect on growth that was seen allows us to conclude that the IGF-1R inhibitory effect is proximal in the pathway and filters down to the entire pathway machinery. Further investigations with this orally available drug need to be performed in the in vivo setting in order to evaluate more completely the efficacy as a human treatment agent, most likely as part of a multimodal treatment approach.
This work validates the role of the IGF axis as a therapeutic target in MPM. The use of NVP-AEW541, other small molecular inhibitors of, or antibodies directed at, the IGF-R are of great potential use in the treatment of this deadly disease, MPM. Evaluation of inhibitor activity on this pathway in MPM should be undertaken and the use of novel agent exploited.
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DR WHITSON: We have looked at 10 different cell lines and four different tissues of malignant pleural mesothelioma. All of those expressed the IGF receptor pathway. The expression of the IGF receptor pathway has been shown in multiple cancers. This particular compound has been evaluated in in vitro work that we have done with just these two cell lines for malignant pleural mesothelioma. It has also been looked at by other institutions in osteosarcoma and fibrosarcomas and solid-tissue tumors in a murine model and also in cell culture. So, the IGF pathway appears to be more involved in several cancers.
DR W. ROY SMYTHE (Temple, TX): I enjoyed your paper. That's a very fine series of experiments that you've done. I have one comment. We actually stained, in my laboratory, about 30 mesothelioma specimens and saw that the IGF receptor was dramatically upregulated in the tumor as compared to normal surrounding lung or pleura. That leads to a question as to what actually is the mechanism for upregulation of this pathway. Is it just simple overexpression of IGFR or is it constitutively activated? Is there a mutation in the IGFR gene in these tumors? Have you looked to see actually what is surrounding it at the cell surface?
DR WHITSON: There are several things there. We have not looked at the actual expression of the protein in a quantitative fashion in this study. In previous work from this laboratory using microarray analysis and PCR, we looked at the copy numbers of the IGF receptor. It was found that compared to nonmalignant mesothelioma cells, the IGF receptor was upregulated or expressed more. There is an autocrine and paracrine effect from the tumors themselves that seems to be an autostimulatory secretion of basal levels of IGF, and then that will feed back on itself. This constant stimulation may lead to the upregulation that is seen.
DR SMYTHE: We have also noted that EGFR [epidermal growth factor receptor] is dramatically upregulated in most of these tumors as compared to in normal surrounding tissue, and something you may want to try as you move into in vivo experiments is maybe working on inhibiting both receptors simultaneously, as EGFR obviously drives a couple of different transcription pathways down to the nucleus. Excellent work.
DR WHITSON: Thank you. That was one of the reasons we used the EGFR receptor and its phosphorylation as a marker for the nonspecific binding. We were concerned that there might be some activation or inhibition of that pathway. But that's a point well taken. We'll look at that as well. Thank you.
DR DANIEL J. BOFFA (Cleveland, OH): You're blocking the downstream interactions at the level of the receptor. Have you looked at why that actually is causing a decrease in the cell number? Is it inducing apoptosis? Is it inhibiting cell cycle progression? Do you have any idea of why, at the end of the day, there are fewer cells in the treatment group as compared to the nontreatment groups?
DR WHITSON: No, we have not looked at that. The only thing that comes to mind as the reason that that might be is if we are inhibiting IGF receptor, even from their own autostimulation, the cells are not activating the IGF pathway. In that fashion, they're not inhibiting the apoptosis, so that regular cycle of the cell would continue. Whether it's a direct toxic effect of the drug or an inhibition of that pathway, we do not know.
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= 0 µM; —x— = 1 µM; -x- = 5 µM; • = 10 µM; — — = 20 µM; 
= 50 µM.)
